Cervical cancer is currently the second most lethal cancer threatening women globally after breast cancer. Each year about 500,000 people are diagnosed as cervical cancer in the world, and about 300,000 people died of this cancer. In China the incidence rate of cervical cancer each year is estimated to be around 135,000 people, and around 50,000 people died of this cancer. It was confirmed in early 1980's that cervical cancer is correlated with human papillomavirus (HPV) infection. Recent data demonstrated that 99.8% of the biopsy samples of cervical cancer were detected to be infected with HPV. Therefore, in care of the reproductive health of women, scientific researchers of various countries have been conducted research on the diagnosis, prevention, and treatment of HPV. It is exciting that the first tetravalent and bivalent HPV prophylactic vaccines (Gardasil and Cervarix) were successfully developed respectively by Merck in the U.S. in 2006 and by GlaxoSmithKline (GSK) in UK in 2007.
However, in terms of HPV prevention and treatment, currently there remain many issues that need to be addressed. It is now clear, for example, that there are 120 of HPV subtypes that can infect the epithelial tissue of female genital tracts, of which approximately 40 are high-risk HPVs (HR-HPVs) resulting in cervical cancer (1-3). The prophylactic HPV vaccines, Gardasil and Cervarix mentioned above, that have been approved for clinical usage in many countries, are mainly aimed only at the most prevalent HPV 16 and 18subtypes, although they also showed a certain degree of protection against other HR-HPVs (4). Therefore, development of “universal” HPV vaccines becomes a widely concerned topic undoubtedly, and they are particularly important to the women in China or Asian, since the HPV58 and HPV52 subtypes are more prevalent than HPV18 after HPV16 among the HR-HPVs that infect women in this region, while HPV18 is usually ranked second after HPV16 in many areas (5).
In addition, both HPV tetravalent vaccine made by Merck and the bivalent vaccine by GlaxoSmithKline have very complicated production processes and very high comprehensive costs, and the prices of these vaccines are obviously not suitable for their promotion and application in developing countries. Thus it is currently an important approach or direction to develop recombinant multiple-epitope peptide vaccines that could be more easily manufactured and stored, and could possess lower prices. Although current HPV therapeutic peptide vaccines mainly select cytotoxic T cell epitope (TCL/TcCE) of the E6 and E7 proteins against HPV 16 and 18 as immunogen, there were reports about the development of therapeutic HPV vaccines of fusion protein that simultaneously induced CD4+, CD8+-mediated and antibody-mediated immune responses by targeting at early carcinogenic E6 and E7 proteins, implicating the possibility of developing prophylactic HPV vaccines of multiple-epitope peptides that combine B- and T-cell epitopes, i.e., achieving the effects of completely or partially removal of HPV by both the humoral response of antibody production and the tumor-specific cellular immune response before the occurrence of precancerous or cervical malignant lesions.
Moreover, in the clinical practice, it is always an important topic to develop a simple, accurate and sensitive serological detection method for HPV infection, a method lying between cytology observation and DNA diagnosis. The core content or technology of the methodology is the identification of antibodies induced by HPVs in the patient's serum as well as the linear antigen epitopes of the induced antibodies (6), followed by the synthesis of the epitope peptides that serve as detection antigen for establishing ELISA detection method. However, so far there has been no a serological detection kit that could be used in clinical diagnosis for HPV infection due to the difficulty in ruling out the high false-positive rates by using the above ELISA detection method, and such a high false-positive rates could not be accepted by both doctors and patients. It is conceivable that the false-positive results are caused by three factors: 1) low titers of antibodies in patients induced by HPV infection, resulting in low extent of dilution of the serum samples (commonly used serum dilutions: 1:2-1:10); 2) there are usually a large number of antibodies against unknown antigens in human serum due to many factors such as its resistance to microbial infections including viruses as well as autoimmunity; 3) take for example to use a single 18˜20-mer epitope peptide as detection antigen (7), it is known that a linear B cell epitope usually comprises of 3 to 8 amino acid (aa) residues, suggesting obviously that there are a large number of potential epitopes composed of more than 3-8 residues on the peptide, and such epitopes can be identified by unknown antibodies. Thus there is a high probability that such a peptide can be recognized by one or several unknown antibodies in the body at lower extent of dilution of the patient's serum. On the basis of the knowledge above, it is believe that in the future in order to develop detection antigens of synthetic peptide with high specificity and sensitivity, the direction for making innovative breakthrough should be: 1) it would be better to make detection antigen comprising the minimal motif of epitope or its extended 8-mer peptide, so that its cross reactions with unknown antibodies would be minimized, thus improving its detection specificity; 2) to make detection antigen peptide comprising the minimal motifs of multiple epitopes as to identify antibodies against multiple epitopes of the target protein. This approach can increase the extent of dilution of the serum samples by enriching the antibodies of lower titer and via the amplifying effect of coupling the second antibody, resulting in the improved detection sensitivity for the target antibodies. However, the non-target antibodies at the same low titer will fall below the detection sensitivity as the extent of dilution of the serum sample increases, thus reducing the potential cross reactivity with the detection antigen and thus avoiding false positive results. Obviously, the success of developing the detection antigen kit described above would rely on the successful identification of more of the linear epitopes of the target antigen as well as their minimal motifs.
In view of the actual needs of developing HPV vaccines and diagnostic reagents, we have previously completed the identification of all four linear epitopes and their minimal motifs of the HPV58-E6 protein (8). We found that the sequence of 84YGDTL88 on the E6-2 epitope of HPV58 is highly conservative among the homologous proteins of the high-risk HPVs (FIG. 1), and this sequence is the same as the five residue sequence at the C-terminal of the HPV18-E6/2 antigenic peptide with 22 aa residues (9).
As is known to all, one antigen epitope produces one kind of antibody, and one antibody corresponds to one epitope. In view of the potential applications of epitopes and antibodies in developing antiviral or antitumor drugs (vaccines) as well as diagnostic reagents, it has no doubt that identification and preparation of epitopes are inventions creating intellectual property rights. Of course, the reason for granting intellectual property rights to such efforts in identification and preparation of epitopes relies on the difficulty of the technology used in the pursuit. Banks L et al., for example, prepared six strains of monoclonal antibodies (mAbs) including C1P5 against recombinant HPV18 E6 protein (10), but did not identify their epitopes and their antibody-recognizing minimal motifs, and thus failed to find that the mAbs C1P5 had cross-reactivity with the homologous proteins of other high-risk HPVs in addition to HPV16-E6 (thus losing other potential applications of C1P5). They also failed to identify the epitopes recognized by the other five strains of mAbs named D2A6, C1N1, C1X1, B1B3 and B1A2. It is also very important to identify the epitopes recognized by the other five mAbs. The subtypes of the five strains of monoclonal antibodies might be different. There are possibilities that the same epitope motif could be recognized by two or more strains of monoclonal antibodies of different subtypes. In another study, for example, two different strains of mAbs with different subtypes (G1 and G3) against HPV18-E6 protein could recognize likewise the four residues motif of 7PTRR10 (11). However, restricted by the methodology of epitope identification, very few researches in this aspect were reported previously. The reason is that the previous technologies of epitope identification including chemical synthetic peptide methodology are difficult to operate, even when the monoclonal antibodies were available to be used for identifying the epitopes.
Therefore, obviously it has great significance in the field to identify the minimal motifs of epitopes of the new monoclonal antibodies by employing our improved biosynthetic peptide method.